CA2912765A1 - A method of determining virus removal from a sample containing a target protein using activated carbon - Google Patents

Abstract

The present invention provides methods for determining whether activated carbon can be used for removing viruses or a certain virus from a sample containing a target protein.

Description

A METHOD OF .DETERMINING VIRUS REMOVAL FROM A SAMPLE
CONTAINING A TARGET PROTEIN USING ACTIVAT.ED CARBON
Field of the Invention 100011 The present invention relates to methods,:of determining Whether activated carbon can be used fbr the removal of virtmes from a sample containing a target protein.
Background

[0002] The most commonly used processes for purifying target proteins such as, e.gõ
monoclonal antibodies, typically employ an engineered cell line (e.g.,: a mammalian or:a:non-mammalian cell IMO:capable of expressing the target protein. Such a target protein may either be secreted into the cell culture media or it may he expressed intracellularly and recovered following Iysis of cells expressing the protein.

[0003] The target protein typically needs to be subjected to:a series of purilicatiOn steps to separate the target protein from various impuritiese,g., tells, cell debris, DNA, host cell proteins etc.

[0004] A typical purification process usually entails subjecting the cell culture feed or media (in case:of a secretory target protein) or cell lysate (hi case of an intracellular target protein) to a variety of steps, including one or more chromatography steps to isolate or purify the target protein. For example, in case of a secretory target protein, monoclonal antibody, the cell culture media is typically subjected. to a clarification step followed by a capture step I-billowed by one or more of a cation exchange bindlelute chromatography Step and an anion exchange chromatography step.

[0005] CH() cells are commonly used for the production of monoclonal antibodies.
A typical CHO cell culture -feed contains 10 to109viruses or virus-like particleS and the removal of such 'viruses or virus-iik.particles is especially important during the pttrification process, as many of the, target; proteins are therapeutic proteins which are directly administered to =patients. Generally, vitus"removal is evaluated based on the amount of total virus removal achieved by the entire purification process, which is desirable to be equal to or exceeding 18 logs especially in eases : where regulatory approval is required.

[0006] Generally, each step of a typical purification process is Shown to remove some amount of viruses, however,, the amount is usually less than 18 logs; and therefOre, additional purification steps must be included in the process for adequate virus removal. It is important to establish whether or not a particular purification step can remove a certain amount of virus in order to ensure that adequate virus rertioval is achieved by the various purification steps in a purification process.

[0007] Guidance for virus safety c.valuation of samples, e.g., biotechnology products, can be found in "Harmonized Tripartate Guideline: Q5A Viral Safety of Biotechnology Products Derived From Cell Lines: of Human or Animal Origin.
Fed, Reg, 63(185) 24 September 1998," prepared under the auspices of the International Conference on Harmonization of Technical Requirenientsk.r Registration of Pharmaceuticals for Human Use (ICH),

[0008] Activated carbon has previously been used in water purification applications to remove viruses as well has been incorporated in filtration units for non-specific removal of substances, which may include viruses, from biological fluids, blood (e.gõ see, US. Patent No. S123.940) 100091 Further, U.S, Patent Application Serial No. 13/565,463, tiling date August 2, 2012, incorporated by reference herein in its entirety, describes the use of activated carbon in combination with other media for removal of proteinaceous impurities (o.g.., host cell proteins) and DNA from a sample containing a blomolecule of interest,(0...g., an antibody).
[00101 Lastly, US. Provisional Patent Application Serial No. 61/769,269, filing date February 26, :2013, incorporated by reference herein, deseribes the use of activated carbon tbr the:selective removal of aprotein from a Mixture of .proteins by changing solution Conditions.
Summary of the Invention [00111 The present invention is based, at least:in part, on the surprising and unexpected discovery that activated carbon can be used for reducing the amount of viruses in ease of certain 8amples containing a target proteit4 e,g.,:a monoclonal antibody. Although, activated carbon has previously been described as removing some viruses during water purification, the samples that are treated with activated carbon generally have a loW concentration ofprotein or no protein at all. The present invention is based, at least in part, on the unexpected and surprising discovery that activated carbon specifically binds viruses, including parvariruses, retroviruses and retrovirus-like particles, in certain samples that include a high protein concentration (e.g., cell culture feed or eel] lysate containing a target protein to be isolated).

Therefore, the inclusion of activated carbon in certain purification processes tan result in the potential elimination of one or more expensive chromatography steps (e.g., a cation exchange bindlelute Chromatography step) that are typically used during protein purification.
[0012] In various embodiments, a method for determining whether activated carbon can be used for the removal of viruses from a sample Containing 3 target protein is provided, where the method coMprising the steps of: (a) providing a portion of the sample comprising a target protein; (b) adding a virus to the portion of the sample in the amount ranging from 104 to 109 PIUME,; (b) flowing the portion of the sample through a column packed with activated carbom(c) collecting one or more fractions containing the target protein from the Column; and (d) measuring the virus amount in the one or more fractions, wherein a reduction in the virus amount in one or more fractions by at least 3,0 1_,RV is indicative that activated carbon can be used for removing viruses from the sample.
[0013[ In some embodiments the sample coMprisesa protein concentration equal to or greater than 0.2 sOrite enabodiments, the sample compri.ses: a pH in the range .of 3.:0- 10.0 and/or a alt. concentration less than 0.5M.
[0014j In some embodiments, the sample comprises:atell culture feed. In a:
particular embodiment, theca culture feed is a CHO cell culture 'feed, [0015] In some embodiments, the sample comprises a cell culture feed that has been subiected=to clarification. Clarification methods include, but are not limited to, centrifugation, settling, depth filtration, screen filtration, flocculation, use of a Stimulus responsive polymer and pH change.
[0016] in sonic embodiments, the sample comprises an cluate recovered from a Protein A chromatography:step used during the purification of the target protein.
[0017] In some embodiments, the sample ictimprises a cell lysate obtained from cells expressing a target protein intracellularly.
[0O18 In some embodiments, the target protein is a recombinant protein:
Exemplary target proteins include, but are not. limited to, immunoglobulins, e.g., a _monoclonal antibody, Fe-containing proteins A$ well as non-immunoglobulin proteins.
[0019] in a particular embodiment, the target protein is a therapeutic protein.
Brief Description of the Drawings [0020] Figure 1 is a graph depicting the results of experiments to determine if a parvoVirus, represented by minute virus:pimice (MVM), Pail be removed from a solution of monoclonal antibody by flowing the solution though a column of activated carbon.. as observed for four different types of monoclonal antibody samples.
MVM
is removed in an amount greater than 5.0 LRV for two separate monoclonal antibody solutions, MAB1 and MABIV, at column loads of 1.0 kg/Land 2.0 kg/L. However, MVM is not removed in an amount above 1.0 LRV for two other monoclonal antibody SaUtions, MABII and MAB1 11. at column loads of 0.5 kg/I,, 1.0 kg/L, and 2.0 kg/L. The X-axis' depicts the amount (in kgs) of the monoclonal antibody that is loaded onto the column per liter (IL) of activated carbon at the point a fraction is taken and the Y-axis depicts the log reduction value of the M-VM virus in the fraction. The upward pointing arrows indicate that the concentration of virus in the sample is below the limits of detection for the fraction.
[0021} Figure 2 is a graph depicting the results of experiments to determine if a retrovirus or retrovirus-like particle, represented by xenotropic marine leukemia virus (XIVIaLV), can be removed from a solution of a monoclonal antibody by flowing though a column of activated carbon, as observed for four different types of monoclonal antibody samples. XMILLV is removed in an amount greater than 3.0 LRV Ibr MARI antibody solution at column loads of 0.5 kg /1õ 1.0 kg/I_ and 1.5 kg/L.
XMuLV is removed in an amount greater than 5.0 LRV at column loads of 0,2 kg/L, 0.5 kg/L, and 1.0 kg/L, in case of antibody solutions, MA1311, MABIII and MAI3fV.
The X-axis depicts the amount (in kgs).of the monoclonal antibody that. is loaded onto the column per liter (L) of activated carbon at the point a fraction is taken and the Y-axis depicts the log reduction value of the IXMitLY virus in the fraction. The upward pointing arrows indicate that the concentration of virus in the sample is below the limits of detection tor the fraction.
Detailed Description [00221 As discussed above, the present invention is based, at least in part, on the novel and unexpected discovery that activated carbon can be used for the removal of viruses from certain high protein concentration samples such as, e.g., certain cell culture feeds or cell lysates comprising a target protein to be purified, [0023.1 The typical concentrations of virus in a bioreactor feed containing a therapeutic protein are generally very low. However, these low levels may still be dangerous to a path:1U being treated with the purified therapeutic protein. In order to ensure adequate removal of viruses during protein purification, it is important to determine whether a certain purification step k01 remove viruses from a sample containing a protein .to be purified and .also determine the amount .,of the virus removed bysuch purification .step, [0.024] In order to do so, a representative virus is spiked into a solution containing a therapeutic protein at a known. concentration and then the virus spiked solution of.
therapeutic protein is be subjected to a certain purification step.õ Following the purification step, the amount of the representative virus in the. solution is determined.
Adequate virus removal by the purification step is indicated by the level of virus being lower than detection limits for that virus or being. undetectable in the solution following the purification step. ParVOVintses and retrOVirttses are two types of .viruses that are .generally measOred during the purification of therapeutic proteins expressed in 0-10 ceUs 100251 Typically,.a method used to determine the amount of virus removed from a sample containing a protein during a purification step employs., aportion of the sample.
instead of the entire .sample Volume; Oncea determination is made that a particular purification step is able to .remove virusesfrom a portion ola sample containing a target protein, it can be assumed that the purification step will remove viruses from the sample, as long as the ratio of the purification .media employed and the target protein are kept the same..
[00261 The present invention. provides Methods for determining .whether activated .carbon can be used for the rernoval of certain viruses from certain samples, .As demonstrated in the: Examples herein,usinu the methods described heroin, it can be determined wheth.eractivated .carbon.is able to remove viruses from a particular sample coniaining.atarget proteih. Therefore, for those samples containing, a.
target protein, where aetivnted Carbon is able. to rernOve.viruseS,activated. carbon can subsequently be incorporated in the purification process: for that sample,.
[0027] In order that the present disclosure may be more readily understood, certain terms are first defined. Additional .definitions are set forth 'throughout the. detailed description.
Definitions MA The. term "active carbon" or"activated carbon," as used interchangeably herein, refers toza carbonaceous material which has been subjected to a process to enhance Its'pOre. structure. Activated carbon i onietinits also referred to as activated charcoal. Activated carbons are porous: solids with very high surface areas:
They can be derived from a Variety of sourceS including coal, wood, coconut husk., nutshells, and peat. Activated carbon can be produced from these materials using physical activation involving heating under a controlled atmosphere or chemical activation using stow acids, bases, Or oxidants. The activation processes produce a porous structure with high surface areas that give activated carbon high capacities for impurity removal. Activation processes can be modified to control the acidity of the surface:
[0029] Typical activation processes involve subjecting a carbon Source, such as, resin wastes, Coal, coal coke, petroleum coke, lignites; polymeric materials., and iiimocellulosie materials including pulp and paper, residueS: from pulp production, wood (lik.e wood chips. Sawdust, and wood flour), nut shell (like almond shell and coconut shell), kernel, and fruit pits (like olive and cherry stones) to a thermal process (e..gõ, with an oxidizing OS) or a chemical proatii- (e.g.,: with phosphoric acid or metal salts, such as zinc chloride). :An exemplary process involving chemical activation of wood-based carbon with phosphoric acid (H3p04) is disclosed in U.S. Patent No.
Re.
31,093, which Tesulted in an improvement in the carbon's decolorizing and gas adsorbing abilities: Also, U.S, Patent N. 5,162,2$6 teaches phosphoric acid activation ofwd-b4ed material which is pal-Ocularly dense:and which contains a relatively high (30%) lignin content, such as nut shell, fruit stone, and kernel.
Phosphoric add activation of lignocellulose material is also discussed in 'U.S. Patent No. 5,204,310, as .a step in preparingtarbons of high activity and high density. The teachings of each of the patents listed in this paragraph are incorporated by reference herein in their entirety.
[00301 in contrast to most other adsorbing :materials:, activated carbon is believed to interact with moleculeS lusitila relatively Weak Van der Waals or London dispersion :RN:Yes. Typical :commercial activated carbon products exhibit a surface area of at least 300 m'ig, as measured by the nitrogen adsorption based Brunauer-Emmett-Tel ler ("BET") method, which is method Well known in the art.
[0031] Although. active or activated carbon has been previously employed in processes for parifYing ii qukisand gaSes as well as for purifying a recombinant!), expressed antibody from other impurities by binding to impurities, it has not been previously employed for removing viruses, especially pan/oviruses and retroviruses, from high protein concentration samples:(Leõ >02 !WI, :prOteiti concentration), Consequently, activated carbon provides a cost effective solution, in: some instances, far the removal of viruses during processes for purifying therapeutic proteins, e.g., monoclonal antibodies.
[0032] The present invention provides methods to determine Whether or not activated carbon can he used for removal of viruses from a. sample containing a target protein.
Because activated carbon does not result in effective removal of viruses: in ease of all samples, based on the methods provided herein, it can be readily determined as to in case of which saMples, activated carbon can be. used for removal of viruses or a particular type of virus, [0033] The terms "Protein of interest" and "target protein,' as used interchangeably herein, refer to a protein or polypeptide, which is to be purified from a composition containing viruses. In some embodiments, the target protein is a therapeutic protein to be administered to.a. patient.
[0034] In general, the .overall purity of the target protein increases as a result of virus removal. The target protein may be an immunoglobulin ot a non-immunoglebulin protein and may be a. secretory protein or an intracellular protein. In some embodiments, the target protein is an immunoglobulin protein, e.g.,. a monoclonal antibody.
[0035] Other examples of target proteins include recombinant proteins which include, hut are not limited to, recombinant burn= growth hOTM011, recombinant human insulin, recombinant follicle-stimulating hormone, recombinant factor VIII (anti-hernophitic factor), recombinant human erythropOietin, recombinant granulocyte colony-stimulatin2: fadtor, recombinant alpha-galactosidase a, recombinant iduronidase; recombinant galsulfase, recombinant dornase alfa, recombinant tissue plasminogen activator, recombinant human interferons, recombinant insulin-like growth factor I, and recombinant. asparaginase, [0036] in other embodiments of this invention; target proteins arc proteins deriVed from human blood or other physiological fluids. Examples of such proteins include, but not limited to, immunoglobulins 0 and M, Factor V1T1., Factor IX, antithrombin and alpha-l-antitrypsinõ
[0037] The term "immunoglobulin,"Ig" or "IgCl" or "antibody" (used interchangeably herein) refers to a protein having a basic four-polypeptide chain structure consisting of two heavy and two light chains, said chains being stabilized, for example, by interchnin disulfide bonds, which has, the ability to specifically bind antigen. The terra "Single-chain immunoglahulie or "Single,-chain antibody"
(Used interchangeably herein) refers to a protein having a two-polypeptide chain structure consisting of a heavy and a light chain, said chains being stabilized, fir example, by interchain peptide linkers, which has the ability to specifically bind antigen. The term "domain" refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (eg., comprising 3 to 4 peptide loops) Stabilized, for example, by pleated sheet andior intraehain disulfide bond. Domains are further referred to herein as "constant" or "variable", based on the relative lack of sequence variation within the domains of various class members in the case of a "constant" domain, or the significant variation within the domains of various class members in the case of a "variable" domain. Antibody or polypeptide "domains" are often referred to interchangeably in the art as antibody or polypeptide "regions". The "constant"
domains of antibody light chains are referred to interchangeably as "light chain constant regions", "light chain constant domains', "CL" regions or "CL"
domains.
The "constant" domains of antibody heavy chains are referred to interchangeably as "heavy chain constant regions", "heavy chain constant domains", "Cfrregions or "CH" domains. The "variable" domains of antibody light chains are referred to interchangeably as "light chain variable regions". "light chain variable domains, "VI:" regions or "YU' domains. The "variable" domains of antibody heavy chains are referred to interchangeably as heavy chain variable regions'', "heavy chain variable domains", "VI-I" regions or "V 1-1" domains.
[00381 Immunoglobulins or antibodies may be monoclonal or polyclonal and may exist in monomeric or polymeric form, ihr example, IgNil antibodies which exist in pentameric form and/or IgA antibodies. which exist in monomeric, dimeric or al U. I till wric form. Immunoglobulins or antibodies may also include multispecific antibodies (e. g., bispecific antibodies).
[003911 The term "Fe region" and -Ftregion containing protein" means that the protein contains heavy and/or .light chain constant regions or domains (CH and CI, regions as defined previously) of an i MUlloglobulin. Proteins containing an "Fe region" can possess the effector functions of an immunoglobulin constant domain.
An "Fe region" such as C1-12/CI-E3 regions, can bind selectively to affinity ligands such as Protein A or functional variants thereof. In some embodiments, an Fe region containing protein specifically binds Protein A or a functional derivative, variant or fragment thereof, In other embodiments, an Fc region containing protein specifically binds Protein or Protein L. or functional derivatives, variantS or fragments thereof.
[0040] As discussed above,. in some embodiments, a target protein is an Fere:Won containing protein,. e. g., an immunoglobulin. In some :embodiments,: an Fe region containing Protein is a recombinant protein which includes the Fe region of an immunoglobulin fused to another polypcptide or a fragment thereof [0041] Generally, an immunoglobulin or antibody is directed against an "antigen" of interest. Preferably,..the antigerniSa biologically important polypeptide. and administration of the antibody lo..annammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal, [0042] The term "monoclonal antibody" or "Mab," as used interchangeably herein., refers to an antibody obtained from apopulation of substantially homogeneous antibodies, Lgn The individual antibodies in the population are identical except for possible naturally occurring mutations that may bepresent in minor amounts.
Monoclonal antibodies are highly specific. being directedagainst a single antigenic.
site. Furthermore, in contrast to. conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants.
(epitopcs), each monoclonal antibody is. directed against a single determinant on the antigen. The modifier. "monoelonal" indicates:the eharacter.of the antibody :as being' obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibodyby any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made.by:the hybridoma method firsudeseribed by Kohleret.ci Nature .256:495 .(1.97.5), Or may be made byrecombinant DNA methods (see,:
e..g.,õ
Patent No...4416,567.).. "Monoclonal antibodieS" may also be isolated from 1)liage antibody libraries using the techniques described in Chic.kson et.a/..> Nature 352:624-628 (1991) and Marks etal.,), Mol. No1_222:581 -597 .(1.991) Monoclonal antibodies may also be referre.d to as "MAbs" or "rnabsr or "mAbs". or "MABs."

[0043] Monoclonal antibodies:May further include "Chimeric": antibodies Cimmunoglohulins) in which a portion of heavy And/or light chain is identical with or homologous to corresponding 'sequences in antibodies derived. from a particular species or belonging.tO apartiettlas antibody.ClassorsubelOs, while the remainder of the chain(s):. is identical with or homologous to corresponding sequences in antibodies.
derived frOth another species or belOnging to.anotherantibOdy :ClaSs.Or subclass, as

9 well as :fragments of such antibodies,: so long as they exhibit the desired biological activity (U.S. Patent No. 49816,567; and MOrri SOO. et al., Proc.,. Natl. Acal =Sci. USA
81:6851-6855(1984)).
[00441 "Humanized" fbrms of non-human (eõg., murine) antibodies are chimeric.
antibodies which cOntain minimal sequence derived from non-human immunoglobulin. For the most. part, humaniied antibodies are human.
immunodobulins (recipient antibody) in which hypervariable region residues of the recipient are roplaced=by hypervariable region residues from a non-human species (donor antibody) such as mouse,: rat, rabbit or nonhuman primate having the desired specificity,. 'affinity, and capacity. In Srarte instanceS,..Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized anti bodies..may comprise residues which are not found. in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. in:general, the humanized antibody wit I
comprise substantially all Of at least one, and typically:NI/O., variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunaglobulin and all or substantially all of the FR regions are those of a, human immunoglobulin. sequence. The humanized antibody may comprise at least:a portion. of an immunoglobulin constantregion (Fe), typically that of a human immunoglobulin. For further details, See Jones el al., Nature 32h522-525 (1986);:.
Riechtnann 0'4, Nature 332:..323-329 (1908);..and Presta, Carr, Op. Struct.
Biol.
.250.-596 (1992).
[0045] The terin'solut.iolf or "sample,".aS Used herein, refers to a composition containing a target. protein which is subjected to virus removal using actiyated carbon, once it is demonstrated using the methods described herein that activatedcarbon can be used for removing viruses .from the composition. in some embodiments, the sample comprises tell culture feed,. tbrexample, feed from a mammalian cell culture gõ.CI-1.0 cells).expressing akeretory .target protein, 04õ a monoclonal antibody.
Insome embodiments, the samplocomprises..a.con lysate.obtained from mammalian or non-mammalian cells ekpresSing:a *argot .protein intraceflutarfy. in Sortie embodiments, the sample comprises a cell culture feed that has been subjected to clarification. in. some ettbodimehts;. the samplecomprises.art eluate from a Protein A
affinity chromatography:01mq_ $amples..also :encompaSs non-mamm.alian expresSion.systeins used for producing a protein:of interestor target protein.

[0046] The tent= "non-mammalian eXpreSsian systems,7 as used herein, refers to all host cells or organisms employed to generate therapeutic proteins, where the host cells or organisms are ofnon-mammalian origin. Examples of non-mammalian e4ression systems used for producing a protein of interest or target protein include yeast such as, Saccharomyeea cerevisiae and Pichler=pastoris, bacteria such as E*chet'khia coIz 11(4111ils meggkriuti4 Brvvibacillus= choshine0,0s, insect cells such as ::.'po4opte.ra:frugiperda cells, Baculovirus infected insect cells, and algae cells.
[0047] 'Then:tin "portion of a sample' refers to part of a sample containing a target protein whichis used to determine whether activated carbon can hewed to remove viruses from the sample; as described herein, tn.general, once it is demonstrated that activated carbon is able t. remove viruses or a certain type of virus from a part of a sample containing a target protein, activated carbon can then be incorporated into a purification process for the target protein in order to remove viruses .or a certain type of virus from the sample,:
[0048] The terms "clarify," '"clarification," and "clarification step," as used herein, refers to a process step for removing:sttspended particles and or colloids, thereby to reduce turbidity, of a :target protein containing solution, as measured in NIL.' (nephelometrie turbidity units) Clarification can he. achieved by a variety of means, including centrifugation or filtration. centrifugation could be done in a batch or continuous mode, while filtration could be donein a normal flow (e.g. depth filtration) or tangential flow mode. In prOcesSes used in the industry today, centrifi.igationis typically followed by depth filtration intended to remove insoluble impurities, which May not flav been removed by centrifugation. Furthermore, Methods for enhancing clarification efficiency can be used, e.g.
precipitation.
Precipitation of impurities can be performed by various meanS.Such as by flocculation, pH adjustment (acid precipitation), temperature shifts, phase change due to stimulus-responsive polymers or:small molecules, or any combinations of these methods. In some embodiments described herein, Clarification involves any combinations of two or more of centrifugation. filtration, depth filtration and precipitation, [0049] The terms "purifying," "increasing the purity," "separating," or "isolating;" as used interchangeably herein, refer to increasing the ratio of target protein to one or more NiNSCS a sample by removing the One or more Viroses from the: sample.
Typically, once it is determined that activated carbon can be used for removing viruses from a sample containing a target protein, as descii bed herein, activated carbon is included in the process for purifying that target protein, thereby to increase the purity Of the target protein.
[00501 The term "virus" Or "viruses" refers to small infectious agents or particles that can replicate only inside a living cell. They are generally composed of nucleic acid (RNA or DNA) enclosed inside a protein capsid, and optionally,=a lipid envelope.
[0051] The term "parvovirttS" refers to linear, non-segmented single-stranded DNA
nonenveloped viruses.; with an aVerage gertOrne Size of 5000 nucleotides and a diameter of 18-26 nm. An exemplary -parrovirus used in the methods described herein is minute: virus of mice.
10052] The term "retroyiruS"irefets:to an RNA Orus which. is capable of integrating its genetic information into the genomic DNA of infected cells via a reverse-transcribed DNA intermediate. The term "retrovirus-like particle" refers to particles generated by cells that have portions of reVovirus-deri yed DNA in their genome.
These particles resemble retroviruses, but are often noninfectious.
.1he:xenotropie aniline leukemia virus, used in the Examples herein, represent a model retrovirus or retrovirus-like particle, [0053] As used herein, the term "remove," "removing," "removal," "reduce,"
"reducing" or "reduction,' as used interchangeably herein, refer to lowering the amount of one or more viruses in a sample wlhich contains a target protein to be purified. As demonstrated herein, activated carbon can be used to remove one or more viruses from wrtuin samples comaininga target protein: to be purified, [0054] The tents "flow-through process," "flow-through mode," and "flow-through chromatography," as used interchangeably herein, refer to a product separation technique in which at least one product in asample is intended to flow through activated carbon (e.g., target protein), while at least :one potential component binds to the activated carbon viruses).
[0055] The sample intended to :flow through is generally referre4to as the "mobile phase' The "flow-through moide"::is generally anisocratic operation (i.e., 4.
process during which the cothpositiOn of the mobile phase is tot changed). The media used fhr flow-through is usually pre-equilibrated m;=ith the same huller solution that contains the target protein molecule. After purification, the media can be flushed with additional quantity of the same buffet to increase the product recovery.

[0056] The term 'tinier" refers to asolution that resists changes in pH by the action of its acid-base corrugate components. Various buffers which can be employed in the methods described herein are described in Buffers. A Guide for the Preparation and IM6 Buffers in Biological :Systems,: QueffrOy, D., ed. Calbiochem Corporation (1975). Different buffers maintain different ranges of pH. for example phosphate buffer is usually used: for pH between 6.0 and 8.0, while for a higher pH, a borate buffer can be used, and for lower pH, a carbonate buffer can be used. Persons of ordinary skill in the art will be able to readily identifY a Suitable buffer to use, depending on the pH to be maintained. Non-limiting examples of buffers that can be used in the methods according to the present invention include IMES, MOPS, MOPSO, Tris, HEVES, phosphate, acetate, citrate, succinate, carbonate, boratei:: and ammonium buffet* as well as combinations of these:
[00571 The term "wash buffer' or "equilibration buffer" are used interchangeably herein, refers to a buffer used to wash or re-equilibrate the actiVated carbon material prior to contacting a sample with the activated carbon.
[00581 The term "conductivity" refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Therefore, With an increasing amount of ions present in the aqueous solution, the solution will have:a higher conductivity:. The unit of measurement for conductivity is milli Siemens per CelitinWter (mSlem or mS), and can be measured using a commercially available conductivity meter (e.g.,: sold by Orion). The conductivity of a solution may be altered:by changing the COTICCIltration of ions therein. Fot example, the concentration of a buffering agent and/or concentration of a salt (e.g. NaCI or l<C1) in the solution may be altered in order to achieve the desired conductivity. Preterably, the salt concentration of the various butThrs is modified to achieve the desired conductivity as in the Examples beloW.
[00591 The term "fraction" or 'fraetiens" refers to a small volume of liquid collected allerstibjecting a sample containing a target protein tO,a purification step, flowing the sample through arvaetivated carbon column. in the methods:
described herein, fractions:are collected from an activated carbon column folio \Ana the loading of the column with a portion of a sample containing a target protein. A
fraction is used to monitor the amount of impurities, such as: viruses, that are passing through the column at a particular point. It i typically expected that the amount Of impurities that pass through a column will increase a$ the amount of the protein passed through the column is increased. The amount of protein in a sample which may be processed by a known amount of material used in a purification step, e.gõ
activated carbon, can be determined by collecting one or more fractions and determining the level of impurities, e.g., viruSeS, removed by that known amount of material.
[0060] The term "column load" or "load: as Used herein, is the value corresponding to the mass of thelarget protein in solution that has passed through a column divided by the volume of the column. The mass of the target protein that has passed through the column is calculated by multiplying the concentration of the target protein in a sample by the volume of sample collected from the column.
[0061] The term "log removal value" or "LIZ \,V".is a value that represents the levels of impurities removed by a column when a known amount of impurity:is passed through. Ltni = lOg(Cfi log(Co, where Cis the .Concentration of impuritieS in the feed solution and Cr, is the. concentration of itnpurities in the feed solution after it has that has passed through the column.
Exemplary Activated Carbon Materials for Use in the Methods Described Herein [00621 Based on the methods described herein which employ activated carbon, it can be determined whether or not activated carbon can be used for the removal of viruses, including parvoviruses and retroviruses, from a sample containing a target protein_ In some embodiments, activated carbon comprises activated charcoal.
Activated carbon can be derived from a variety of sources including, but not limited to, coal, wood, coconut husk, nutshells; and peat. ActiVated carbon can be produced from these materials by physical aetivation involving heat under a controlled atmosphere or by Chemical activation Using strong acids, bases, or oxidants.
The activation processes .produce a porous structure with a high surface area that gives activated carbon a greater capacity for impurity reinoval, Activation processes can be modified to control the acidity of the surface:
10063] Activated carbon is available from a wide variety of commercial sources and comes in a number of grades and formats. Some Of the coMmereial suppliers of activated carbon include companies such as MeadWestYaco COrp., Richmond, VA, USA; Nitwit Americas inc.õ Marshall, TX, USA; Calgon Carbon Corp,, Pittsburgh, PA, USA.

[0064] In some embodiments described herai, activated carbon is incorporated in a cellulose-containing fibrous media.
[0065] Commercially available activated carbon materials that may be employed in the methods according to the present invention include, but are not limited to, Nuchar .14D activated carbon (MeadWestVaco= Corporation, Richmond, VA, USA); Nuchar SA 20 (MeadWestVaco Corporation, Richmond, VA, USA); Nuchar SN
(MeadWestVaco Corporation, Richmond, VA, USA); Nuchar WV-B 30 (MeadWestVaco Corporation. Richmond, VA, USA); ROC Powder activated carbon (MeadWestVaco Corporation, Richmond, VA, USA); Norit Darco KB-G activated carbon (Norit Americas Inc., Marshall, Texas, USA); Norit COP Super activated carbon (-MAU Americas Inc., Marshall, Texas, USA); Norit A Supra USP (Norit Americas Inc., Marshall, Texas, USA); Norit:E Supra USP (Norit Americas Inc., Marshall, Texas, USA); Norit C GRAN (Norit Americas inc,, Marshallõ Texas, USA);
Norit SX Ultra (Norit Americas Inc., Marshall, Texas, USA); and Chemviron Pulsorb PGC activated carbon (Chemviron Carbon, Feluy, Belgium).
[0066] Two major formats of activated carbon are powdered and granular.
Powdered activated carbon contains small and usually less than 1 mm diameter particles, and is most commonly used for purification of liquids, Granular activated carbon has :a larger particle size and consequently a smaller surface area, so it is preferred for use in gas purification Where the rate of diffusion is faster, [0067] An important consideration for safety with use of activated carbon in consumer applications (such as water, food, beverage, and pharmaceutical purification) is reduction and control of extractable compounds. Activated carbon intended for drinking water and food contact applications is usually made in compliance with safety standard ANSUNSF Standard 61 that covers all indirect additives to water. Also, ASTM standard test method D6385 describes determining acid extractable COMM aativated carbon by ashing and could be used to study and the level of extractables from activated carbon.
[0068] A range of activated carbon types is available for various applications. For example, MeadWestVaco Corp. supplies at least twelve types of powdered activated carbon that vary by their capacity, surface acidity, pore accessibility to target molecules, and intended application. It is generally desirable to maximin the capacity of adivated carbon for impurity removal, Exemphini Viruses Used in the Methods Described herein 100691 Exemplary viruses that may be used in the methods desc.ribed herein, include but are not limited:to, Adenovirus, Calicivirus. Transmissible Gastroenteritis Virus, Bovine Diarrhea Virus, West Nile Vitus, Duck Hepatitis B Virus, Cytomegalovirus, Herpes Simplex Virus, Infectious Bovine Rhinotracheitis Virus, Pseudorabies Virus, Influenza Virus, Simian Virus Type 40, Parainfluenza Virus, Human Parvovirus, Minute Virus of Mice, Porcine Parvovirus, Canine Parvovirus, Encephalomyocarditis Virus, Hepatitis A 'VituS, Poliovirus, Bovine Enterovirus, Porcine Enterovitus, Va.ceinia Virusõ geovirus, Mlarine Leukemia Virus (Xenotropic, Amphotropic, Ecotropic). Human immunodeficiency Virus; Vesicular Stomatitis Virus, Sindbis Virus and Semliki Forest Virus.
[0070] A virus is added to a portion of a sample containing a target protein in the amount ranging from 104 to 108 PFUtra. or TCID50,/mL prior to the portion of sample being contacted with activated carbon, as:described herein.
[00711 In some embodiments, the minute virus Of mice; which represents a parvovirus, is added to a portion of sample containing a target protein, In other embodiments, the xenotropic murine leukemia virus, which representS retrovirus or retrOVirUS-like particle, is added to a portion of sample containing a target protein.
IV. Flow-through Processes Using Activated Carbon [00721 One general flow-through procedure Wihich may be used for determining the removal of viruses using activated carbon is:deScribed below.
[0073] In some embodiments, o chromatography device, e.g., column, is loaded with an aqueous slurry of activated carbon. Activated carbon can also be loaded into a device, e.g., a column, as a dry powder and then wetted with an aqueous solution.
However, sometimes it may be challenging to remove ginail air bubbles from in between the activated earborkparticles when the column is dry packed. The column is then equilibrated with..a buffer having the same pH: as the sample containing the target protein. The sample is subsequently passed through the activated carbon Column at a flow-rate that results in a col UM resideme time of between 15 secs and 10.0 mins.:
The eluate containing the target protein is s bsequently collected.
V. Methods of Measuring the Amount of 'Viruses in Samples [00741 Ome portionof sample is treated with activated carbon, as described herein, the amount of virtiS remaining in the sample is measured using one or more methods described herein or those known in the art. These methods include, but are not limited to, tissue Culture infectiouS:dOse (TCI)50) asSay, plaque-forming unit assay, focus,forming unit assay, 50%: lethal dose assay,hemagglutination assay, florescent focus assay (FFA), bicinchoninic acid .assay (BCA), single radial immunodiffusion assay (SRID), quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELBA), transmission electron microscopy.
Generally, the method that is used would be dependent on the virus that is being measured or detected. Some of the assays which may be incorporated in the methods described herein are described below.
[0075] 50% tissue culture infective dose CICID50) is an endpoint dilution assay which quantifies the amount of :virus required to kill 50% of infected host cells or to produce Cytopathic effect in 50% Of inoculated tissue culture cells. Following incubation, the percentage of cell death (Le, infected cells) is manually observed and recorded for each virus dilution and the results are used to mathematically calculate the -rcipso.
[0076] Viral plaque-forming unit assays:determine the number of plaque forming units (PFUs) in a sample by spreading various dilutions of the sample across confluent monolayers of host cells, and then, after a suitable incubation period (generally. 3-14 days), counting the number Of plaques in the monolayer.
100771 A 5" lethal dose (LD50) assay is conducted by administering dilutions of sample into suitable host animals to determine the dose that is sufficient to kill 50%of the animals.
1007.81 The heinagglutination assay may be used to quantify certain viruses.(e,g.
influenza) by incubating serial dilutions of the sample with a 1% erythrocyte solution for one hour and then visually determining the virus dilution at which agglutination first occurs.
[0079:1 Flitorescent focus assays are .conducted similarly to plaque-forming unit assays; except that the number of infectious units is determined by staining the cell monolayer with fluorescent antibodies that specifically bind to: virus components.
:Fluorescence microscopy is used. to count and quantify how many cells are infected.
[00801 A bieinchoninic acid assay (BCA) quantifies virus by measurement of the total amount of protein in a smple. BCA reagent: is, added to the sample, and a protein's peptide bonds quantitatively reduce Cu to Cu'*, which produces a light blue colOn BCAchelat.esCu at a2t I ratio resulting in a more intensely colored species that absorbs at 562 nm. Alpsorbance Of. a Sample at 56,2 pm is used to determine the bulk protein concentration in the sample. Assay results arecompared with known standard curves after analysis with :a spectrophotometer or plate reader.
100811 Single radial immunodiffusion assay (SIZED), also known as the Mancini method.: is a protein assay that detects the amount:1)f Sped& viral antigen by immunodiffusion in a semi-solid medium (e.g. agar): The medium contains an antiserum specific to the antigen of interest and the antigen is placed in the center of the disc. As the antigen diffuses into the medium it creates a precipitate ring that grows until a equilihriUM is reached. .Assay tiine can range from 10 hours EQ
Several days depending on the equilibration time of the antigen and antibody. The zone diameter from the ring is linearly related to the log of protein concentration and it compared to:zone diameters for known protein Standards for quantification.
100821 Quantitative poiymerase chain reaction (AKA.) is a technique that measures the amount of specific nucleic acid sequences in a: Sample. By using probes specific for viral genetic sequences, the number of viral genom.es in a sample may be counted.
[00831 Transmission electron microscopy (TEM) may also be used to quantify virus by generation of high resolution magnified images Of the sample. 'ITEM images can show individual virus particles and quantitative image analysis can be used to determine virus concentration.
[0084] This invention, is further illustrated by the following examples:'which should not be cOnstrued as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference.
Examples Example 1. The production of Minute Virus of Mice [0085.1 This example describes the process tO prepare minute virus of mice (MVM), which represents a paryoyirus. The WM is subsequently spiked into solutions of MAB and its selective removal by activated carbon is examined.
[0086.1 High:titer MVM is generated by infectin4confluent A9 cells (ATCC
CCL-1.4) in Advanced/F12 pulbeeco's Modified Eagle's: Medium (DMF.M) In V ittOpil (Catalog no. 12634,028) containing 1% (WO fetal bovine serum (MS) With penicillin streptomycin (0.2 gg/alL), and 2.0 ruM ',glutamine. Following incubation at 37 'C. and 5% carbon dioxide for 3 days; the media is replaced with the same rnedium containing no serum and infection is Continued for 7 more days, [0087] Cell lysates are harvested and clarified by centrifugation (300g for 20 minutes) and subsequently concentrated by ultrafiltration (EMI) Millipore Corporation, Catalog no. UFC710008). Final concentration is obtained by ultracentrifugation for 4 hours at 112,000 g using an SW28 rotor in a Beckman Centrifuge. Virus pellets are resuspended in TNE buffer (10mM Tris, 100 ritM
'NaC1, 1mM IFDTA at 7.5) befbre a final polishing step using flow through cation exchange chromatography. Purified virus is stored in buffer at -80 9C: for use in spiking studies.
Example 2. Determination of Minute Virus of Mice concentrations' [0088] This example describes the process to determine the amount of minute virus of mice (WIVNI) ina solution. This process can be used to determine how much MNIM
spiked into solutions of MAB remains after treatment by activated carbon.
[0089] MVM titers are determined using a tissue culture infectious doSe 50%
(TO D50) assay as described in Bolton et al., Biotechnol. Appl. Biochem. Vol.
42, 2005, 133-142. Test samples are diluted to mitigate cytotoxicity and viral interference followed by 10-fold serial dilutions prepared with cell culture medium and 100 ;.1.1 aliquots of each dilution are added to each of 16 wells of a 96-well microtiter plate containing sub-confluent 324K. PT cells (obtained from Professor P.
Tattersall, Department of Laboratory Medicine and Genetics, Yale University School of Medicine, New Haven, CT, USA). Following incubation at 37 in 5% CO2 for

10-12 days, infected wells are visually assessed for cytopathic effect (CPE) and titers are determined using Spearman-Karber methods (Spearman, C, British Journal of Psychology, Vol 2. 1908, 227-242; Karber, G, Arch. Exp. Pathol. Pharmak. Vol 162, 1931, 480-483). Where low counts are expected (filtrate samples), large volume plating techniques lare also used. Depending on the frequency of observed CPE, titer of these samples is estimated using one of several different accepted statistical methods. Log reduction values (LRVs) are calculated by determining the log 10 of the viral load upstream and subtracting the log 10 of the total virus in the filtrate, Example 3. The production of Xenotropic Murine leukemia virus [0090] This example describes the process to prepare xenotropie murine leukemia Virus. (XMul..V), which represents retrovirtts or retrovirus-like particles.
The XIMuI, V
is subsequently spiked into solutions of MAB and its selective removal by activated carbon is examined.

[0091] Highly purified XMIIIN is produced by infecting a single flask of MV1Lti cells...(Mink lung cells ATCC.C.C.L-6-4) in the presence of DMEM supplemented with 1.0%:(v/V).letal bovine serum (HIS). with penicillin (Ui Units/ML), streptomycin (0.2 uglin.L.), and 2 tnM ',glutamine. This infected flask .of cells undergoes repeated passaging and expansion in DMEM supplemented with 23% (viv) fetal bovine serum (F.BS) with penicillin (02 Units/m1),. Streptomycin (0.2 lag/W). and 2 niM.
glutamine. The cell culture fluid of infected monolayersis harvested and the flasks are fed with Advanced/F12 Dulbece(V.S.Modified Eagle's Medium (Ad' OME.M) vitrogen.(catalo a. no. I 2634-028.) Containing penicillin (0,2 Units/la), streptomycin (0,2 RgirriL), and 2 mM L-glutamineõ Following incubation at 37."C:
5%.0O2:.fOr2.dayS,:the cell culture fluid.of infected .monolayers is harvested and the monolayers are fed again with Adv DIMEM with penicillin (0.2 Units/mL), streptomycin (0,2 uglinI.,), and 2 triM L-glutamineõ
00921 Following incubation at 37 cc and .5% carbon dioxide for 2 days, the cell culture fluid is harvested. All cell culture fluids are clarified by low speed centrifilgatiOrt.(300.gfor .20 minuteS), filtered through 0.454/11 Durapore filter and purified by eentrifugation at 9500,>:<gfor 2 hours using GSA rotor in Sorvall centrifuge. Pelleted virus is resuspended in protein-free storage buffer and.
stored. at -80 C until ready for use in spiking studies.
Example 4. Determination of Xenotropie Murine leukemia virus concentrations [0093] This example describes the process:4) determine the amount of xenotropie murine leukernia..*ints...(X.MuliN) inasolution. ThisproCessiS: Used to determine how much XMul...V spiked into solutions of MAB remains after treatment with activated carbon.
[00941 XiMuLAT titers are determined using a. tissue culture infectious dose 50%
(17CID.50) assay a.s :described in .Bolton et at, Rioter:111101. Appl.
Biochem. VOL 42, 2005, 1.33:442. TeStSarnples.are diluted.tomitigate:eytotOkidityand viral interference.
-followed by preparation of I 0-1-bld serial, dilutions with cell culture medium, and 1.00 ul aliquots of each dilution are added. to eachof 16wells of a 9.6.-well microtiter plate containing sub-confluent PG4 cells. (ATC..C.C.R1,2032). The platesare.then spinoculated in eentrifitge:(1800 rprn.for 60 mins)._ Following incubation at 37Ce in 5% CO2 for 7 days, infected wells are viSually a*gse4fot=cytopatliie effect fC.PE) and titers are determined using the' Spearman-K'Arber methods .(Spearman,. C..
British Journal of Psychology, Vol 2, 1908, 227-242; Karber, G.:, Arch, E.xp, Pathol.
Pharmak. Vol 162, 1931, 480-483). Where low counts are expected (filtrate samples), large volume plating techniques are also used. Depending on the frequency of observed C.PEõ titer of thew samples is estimated using one of seVeral different accepted statistical methods. Log reduction values (E,R V) is calculated by determining the log 10 of the viral load upstream and subtracting the log 10 of the total virtis in the filtrate.
Example 5. Method to determine whether activated carbon can be used to selectively remove a parvovirus from a MARI monoclonal antibody solution [0095] This is a representatiVe Ocample that demonstrates a method to determine if a parvovirus. represented by minute virus Of mice (MVM), is removed from a solution containing a monoclonal antibody (i:eõ MABI) by flowing through a column of activated carbon. The results indicate that activated carbon can be used to selectively remove a parvovirus from a MABI monoclonal antibody solution.
[0096] A solution of:MA.13f is spiked with MVM and then flowed through an activated carbon column, as described below.
[0097] MARI produced from CHO:tell is clarified and subsequently subjected to Protein A affinity chromatography (eluted with 25 mM acetic acid, 25 raM
glycine HC1). The pH of the Protein A eluate is adjusted to pH 7,0 with 1 M Tris base and the eluate is filtered through aStericup-GP 0.22 pm Millipore Express PLUS:Membrane (IL. catalogue number: :SCCiPii02RE, EMD Millipore Corporation, Billerica, MA, 01821, USA). MARI feed at pH 7,0 is spiked with Ultrapure grade of MVM that is produced aCeording to the procedure described in Example 1 to: target amount of 2.0 E+06 IC1D50/mI,. The spiked feed is filtered through a0.22 um GP express filter before contact with activated carbon.
[00981 A glass chromatography column (011inifit Benchmark Column 10 mm/100 itml, 10 mm diameter, 100 mm length, %CU: 006BCC,10-10-AF, Diba Industries, Danbury, CT 06810, US) IS loaded with 200 mg ofNuchar HI) activated carbon (MeadWestVaco Corporation. Richmond, VA, USA) slurried in water. The column is packed by flowinz water through it, which r6Ailts in a packed column volume of 0,8 rniõ.
[0099j The activated carbon colunut is pre-equilibrated with about 10 CV
of 25 truM iris at pH 7,0: Vitus spiked MAR feed is pumped through the activated carbon column at mUmin (0.8.0//min) with Watson Marlow cassette pumps. 2 ml fractions are: collected from the effluent at column toads of 1,0 kg/L and 2,0 kg/L.
The sample's are titrated by virus infectivity assay. as described in Example 2, As summarized in Table I and Figure 1, this experiment demonstrates that greater than 5.0 LIZN/ of a parvovirus, represented by MVM. are removed from a solution of MABI by flowing the solution though a column of activated carbon at column loads of 1.0 kg/L and 2.0 kg/L. Accordingly, activated carbon can be used to selectively remove a parvovirus from a solution containing MARL
Table LRV of mym removed from MABI containing solution,:measured in fractions collected at various column loads of MARl on the activated carbon column.
The experiment is run in duplicate and the value listed is an average of those two values. Note that the concentration of MVM in the fractions is below the levels of detection.
Column load of MABI
LRV of MVM from on activated carbon MABI
(kg/L) 1.0 >5.03 2.0 ?5.13 Example 6. Method to determine whether activated carbon can be used to selectively remove a parvovirus from a MARLI monoclonal antibody solution [001001 This:is a second representative example along with Example 5 that demonstrates a method to determine if a parvovirus, represented by minute virus of mice (MVM), is removed froth a solution containing monoclonal antibody MARE') by flog through a column of activated carbon. The results indicate that activated carbon cannot he used for selectively removing a parvovirus from a MARII
monoclonal antibody solution.
[001011 A solution of MABTI is spiked with MVM and subsequently flowed throueh an aCtiVated carbon eOltonn, described below.
[001021 MABII produced from CHO cells clarified and then subjected to Protein A column chromatography (e/uted iAdth 25 miM. acetie:acid, 25 mMglycine FICI).
The pH of the Protein A :elttate is adjusted to pH 7,0 with i M Tris base and filtered through a Stericup-GP 0.272 p.m :Millipore Express PLUS membrane (1 L9 catalogue number; S'..(7CliPt_l02R.E, ENID Millipore Corporation, Billerica, MA, 0182/, USA).
The teed is dialyzed intO 25 inN1 Iris pli 7.0 twice with dialysis tubing (Standard RC
Dialysis Thal KitS, Spectra/Por 1.3, 3.5K N1 WCO, 54 min FLAT WIDTH, serial number: 132725, Spectrum Laboratories, Inc. Rancho Dominguez, CA, 90.220 USA).

The solution is then filtered through .a. Stericup-GP 0.22 4111 Millipore Express PLUS
membrane (1 L, catalogue number::SCGPITO2RE,EMD Millipore Corporation., Billerica, MA, 01821, USA). The dialyZed MABil teed at pH 7.0 is spiked with Ultrapure grade of MVM that is produced according to the procedure described in Example 1 to a target amount of2.0 E 06 TODmtmL. The spiked feed is then filtered through a 0.22 uni GP express filter before ColaCtirM with. activated carbon.
[00103] A glass. chromatography column (017111ffit Benchmark Column 10 min1100 mm, 10 mm diameter, 100 mm length, SKUt 00613CC- I 0-10-AF, Diba Industries, Danbury, (2.T 06810, ITS) is loaded with 200 mg of Nuchar HD activated carbon (MeadWeStVaCo Corporation, Richmond, VA, USA). slurried in water. The column is packed by flowing water through it, which results in a packed column volume of 0.8 [00104] The activated carbon column is pre-equilibrated with about 10 CV of .25 mM iris at pH 7,0, Virus spiked mAb feed is pumped through the activated carbon column at 1 mUrnin (0.8 CV/min) With Watson Marto* cassette pumps. 2 ml fractions are collected from the effluent at column loads of 0.5 kg/L, 1.0 kg/L, and 2.0 kg/L.: The samples are titrated using a virus infectivity assay, as described in Fxample 2.
[001051 As summarized in Table ft and Figure 1, this experiment demonstrates that less than 1.0 E.R.V ofpatVOVirus-like particleS, represented by Minute Virus of Mice (MVNI), are removed from a solution of MABil by flowing the solution though .a column of activated carbon at column loads of 0.5 kg/L, 1.0 kg/L and 2.0 kg/L.
The results indicate that activated carbon cannot be used to Selectively remove a parvovirus from a NIABII monoclonal antibody solution.

Table El. LR.V of MVM removed from MABIl in fractions collected at various column loads of MABII on the activated carbon. column.
Column load of MABII
LRV of MV M from on activated carbon MABII
(kg/L ) 0.5 0.96 1.0 0.90 2.0 0.52 Example 7. Method to determine whether activated carbon can be used to selectively remove a parvovirus from a M.ABIll monoclonal antibody solution [001(161 This is a third representative example along with Example 5 and Example 6 that demonstrates a method to determine if a parvovirus, represented by MVIVI, is removed from a solution containing a monoclonal antibody (Le., MABIl I) by flowing through a column of activated carbon. The results indicate that activated carbon cannot be used to selectively remove a parvOvirus from a MABUI monoclonal antibody solution.
100107] A solution of MABI H. is spiked with MVM and then flowed through a column of activated carbon, as described below.
[00108] MABIII is provided as a clarified cell culture by Merck Serono. MA BM
is captured with Protein A chromatography (eluting with 20 mM glycine-hydrochloride butler at pH 2.4 The pH of the MABIII eluate is acUusted to pH 7,0 with 1 NI
Tris base and the solution is then filtered through a Stericup-GP 0.22 pm Millipore Express PLUS membrane (1 L, catalogue number: SCCIPUO2RE, EMD Millipore Corporation, Billerica, MA, 01821, USA). The feed is dialyzed into 25 rnM iris pH
7.0 twice with dialysis tubing (Standard RC Dialysis Trial Kits., Spectra:Tor 1-3, 3.5K
MWCO, 54 mm FLAT WIDTH, serial number:112125, Spectrum Laboratories, Inc.
Rancho Dominguez, CA, 90220 USA). The solution is subsequently filtered through a Stericup-GP 022 urn Millipore Express PLUS menibrane(1 L, catalogue number:
SCGPUO2RE, EMD Millipore Corporation, Billerica, MA, 01821, USA).
[001091 MABI if feed at pH 7.0 is spiked with 1. lt.rttpure grade of MV M that is produced according to the procedure described in Example 1, to a target spike of 2,0E+06 TCID50/ML. The spiked feed is then filtered through a 0.22 pm (..4PexpresS
filter beforecontacting with activated carbon.
1001101 A glass chromatography column (Offinifit Benchmark Column 10 Mtn/ 1 00 mm, 10 mm diameter, 100 mm length, SKU: 006BCC710-10-AF, Diba Industries, Danbury, CT 06810, US) is loaded with 200 mg of Nuchar ETD activated carbon (MeadWestVaco Corporation, Rictimond, VA, USA) slurried in water. The column is packed by !lowing water through it, which resulted in a packed column volume of 0.8 1001I11 The activated carbon column is pre-equilibrated with about 10 CV 025:
niM Tins at pFJ 7:0. Virus spiked MAB feed is pumped through the activated carbon column at 1 mL/min (0.8 CV/min) With Watson Marlowtamette pumps. 2 ml fractions are collected from the effluent at column loads of 0.5 1.0 kg/L., and 2.0 kg/L. The samples are titrated using a virus infectivity assay, as described in Example [001121 Asstitntriartied in Table 111 and Figure 1, this experiment demonstratesilhat less than 1.0 LIZA/ of parvovirus-like particles, represented by Minute Virus of Mice (MVM), are removed from a solution of MABII1 by flowing the solution though a column of activated carbon at column loads of 0.5 kg/Iõ 1.0 kg/L and 2,0 kgIL.
The results of this method indicate that activated carbon cannot he used to selectively remOVe parvovinis-like particles from a MABIll monoclonal antibody solution, Table Ill. LRY.of MVM removed from MA.BIII in fractions collected at various column loads of MAME' on an activated carbon column.
Column load of MABIII
1_,RV OIMVM
on activated carbon from (k.Q1L) 0.5 0.15 1.0 0.15 2.0 0.15 Example 8. Method to determine whether activated carbon can he used to selectively remove a parvovirus from a MABIV monoclonal antibody solution [00113] This is aforth representative example along with Ex:ample: 5, Example 6, and Example 7 that demonstrates triethOd to Op-tern-line if a parvovitus, represented by [AVM, is removed from a solution containing a monoclonal antibody (i.e.;
MABI bYlIONVing. through a column of activated carbon. The results indicate that activated carbon can be used to selectively remove a parvovirus from a MA 13[V

monoclonal antibody solution.
[00114] A solution of MABIV is spiked with WM and then flowed through a column of activated carbon, as described below, [00115] MABI V monoclonal antibody 1.5 obtained from Merck Serono Biodevelopmentas: a 10 g/L aqueous solution containing 10 mIg. citric acid, 100 inM
*tine, 100 rnM sodium chloride, and 0Ø1% Tweet. The feed is dialyzed into 25 niM Tris pH 7.0 twice with dialysis tubing (Standard RC Dialysis Trial Kits, SpectralPor 1-3, 3.5K MWCO, 54 mm FLAT' WIDTH,: serial number: 132725;
Spectrum Laboratories Inc., Rancho Dominguez, CA, 90220 USA). The solution is filtered through a Stericup-GP 0.22 pin Millipore: Express PLUS membrane (I L, catalogue number: :SCGPUO2RE, EMD Millipore Corporation, Billerica, MA, 01821.

USA). MAB1V feed at pH 7.0 isi spiked with Ultrapure grade of MVM that is produced according to the procedure described in Example 1 to a target spike of 2,0 E406 TC ID50/mL. The spiked feed is then filtered through a 0.22: iam GP
express:
filter before contacting with activated carbon.
[00116] A glasS chromatography column (Omnifit Benchmark. Column 10 trun1100 mm, 10 mm diameter, 100 mm length. SKU: 006BCC710-10-AF, Diba Industries, Danbury, CI 06810, US) IS loaded with 200 mg- ofNuchar HD activated carbon (MeadWes'iVaco Corporation. Richmond; VA, USA) slurried in water. The column is packed by flowing water through it,:which results in a packed column volume of 0.8 [00117] The activated carbon column is then pre-equilibrated with about 10 CV
of 25 mM Tris at pH 7Ø Virus spiked MAB feed is pumped through the activated carbon column at 1 mElmin (0.$ CV/min) with Watson Marlow cassette pumps. 2 ml fractions are caected from theeffluent at column loads of 1.0 kg/L and 2.0 kg/L. The samples :are tinated Using a virus infectivity assay, as described in Example 2.
[00118] As swnmarized in Table IV and Figure 1, this experiment demonstrates that greater than 5.0 1:RV of a patvovirus, represented by MVM, is removed from a solution of MA13.1N7 by flowing the solution though a column of activated carbon:at column loads of 1.0 kg/1., and 10 kg/L. The results indicate that activated carbon can be used tO selectively remove a parvo virus horna MABIV monoclonal antibody solution.
Table IV. LRV of MVM removed from MABIV in fractions collected at various column loads of MARRY on the activated carbon column. The experiment was run in duplicate and the value listed is an average of those two values. Note that the concentration of MVM in the fractions is below the levels of detection.
Column load of MAIRIV
LIZAl of MVM
on activated carbon from MABIV
(IKg/L) 1.0 2.0 >5.38 Example 9. Method to determine whether activated carbon can be used to selectively remove retroviruses and/or retrovirus-like particles from a M AM monoclonal antibody solution [00119] This is a representative example that demonstrates a method to determine if retroviruses and/or retmvirus-like particles, represented by xenotropic murine leukemia virus (XMuLV), arc removed from a solution containing monoclonal antibody (i.e.. MAK) by flowing through a. Whillin of activated carbon. The results indicate that activated carbon can be used to selectively remove retroviruses and retroVirus-like particles from a MABI monocional antibody solution.
[00120] A solution of M AB 1 is spiked with XMaLV and flowed through a column of activated carbon, as described below.
[00121] MAB1 produced in CHO cells is clarified and then isolated using Protein A
column chromatography (elute(' with 25 niM acetic acid, 25 niM gl)cinel-ICI).
The pH of the Protein A elutate is adjusted to pH 7A) with 1 M Tris base and the solution is filtered through a Stericup-GP 0:22 pm Millipore Express PLUS membrane 0 L, catalogue number: SCGPIJO2RE, EMD Millipore Corporation, Billerica, MA, 01821, USA). MABI feed at pH 7.0 is spiked with T FP purified XkluLV that is produced according to the procedure described in &ample 3 to a target spike of 1.0E+05 TVID50/m.L. The spiked feed is filtered through a0.45 um Durapore filter before contacting with activated carbon.
[00122] A glass chromatography column (Onfit Benchmark Column 10 mm/100 mm, 1 0 mm diameter. 100 mm length. SI( 00613CC-10-10-AF, Diba Industries, Danbury, CT 06810, US) is loaded with 200 mg of Nuchar HD activated carbon (MeadWestVaeo Corporation, Richmond, VA, USA) slurried in water. The column is packed by flowing water through it, which results in a packed column volume of 0.8 [00123j The activated carbon column was is pre-equilibrated with about 10 CV
of 25 mr0 Tris at pH 7,0. VirUsspiked MAI3 feed is pumped through the activated carbon column at 1 InLimin(O..8 CV1,..'thin) with Watson Marlow cassette pumps. 2 ml fractions are collected from the effluent at column loads of 0.5 kg/Lõ 1.0 kg/L, and 1.5 kg/L. The samples are titrated using a virus infedivity assay as': described in Example 4, [00124] As summarized in Table V and Figure 2, this experiment demonstrates that greater than 3,0 LRV of retroviruses and retrovirus-like particles, represented by XkluLV, are removed from a solution of MAR( by flowing the solution though a column of activated carbon at column loads of 05 kg/L, 1.0 kg/L, and 1.5 kg/L.
The results indicate that activated carbon can be used to selectively remove retrovirus-like particles from a :MAIM Monoclonal tibody Table V. LRV of NivluLV reinoyed from MABI in fractions collected at various column loads of MAB1 on an activated carbon column. The experiment Was run in duplicate and the value listedis an.: average Of those two values Note that the concentration of X MuLV in the fractions is below the levels of detection.
Column load of MABI LRV of X.MuLV
on activated carbon from MA.B
0<wp 0.5 ?.1,85 1.0 >1.8.5 1.5 >3.85 Example 10. Method to determine whether activated carbon can be used to selectively remove retrovirueses and/or ret rovirus-iike particles from a MABH monoclonal antibody solution [001251 This is a second representative example along with Example 9 that demonstrates a method to determine if retroviruseS and retrovitus-like particles;
represented by XMuIN, are removed from a solution containing monoclonal antibody (i.e.. MASH) by flowing through a column of activated carbon. The results indicate that activated carbon can be used to selectively rein0Ve retroviruses and retrovirus-like particles from a MABII monoclonal antibody solution.
[001261 A solution of MABI I is spiked with NIMAN and flowed through a column of activated carbon, as described below.
[00] 271 MAIM produced in CH() cell is clarified and then isolated using Protein A
column chromatography (eluted with 25 mM acetic: acid, 25 mM glycine HO). The pH of the Protein A eluateis adjusted to pH 7.0 with 1 M Tris base and then the solution is altered through a Stericup-GP 0.22 1.tmiVillipore Express PLUS
membrane (1 L, Catalogue number: SCGPIJO2RE, EMD Millipore Corporation, Billerica, MA, 01821, USA). The feed is sUbsequently dialyzed into 25 mM Tris pH
7.0 twice with dialysis tubing (Standard RC Dialysis Trial Kits. Spectra/Pot' 1-33..5K
MWCO, 54 mm FLAT WIDTH, ;serial number: 132725, Spectrum Laboratories Mc., Rancho Dominguez CA, 90220 USA). The solution is filtered through a Sterieup-GP
0.22 pm Millipore Express PIX.S. membrane (I L, catalogue number: SCGPUO2RE, EMD Millipore Corporation, Billerica. MA, 01821, USA).
[00128] MABII feed at pH IA) is spiked with Ultrapure grade ofXMuLV that is produced according to the procedure described in Example:3 toa target spike of 1,0E+06 ICED50/ML. The spiked teed is filtered through a 0.45 um Durapore filter before:contacting with activated carbon, [00129] A glass chromatography column Wirmitit Benchmark Column 10 mm/100 mm, 10 mm diameter, 100 mm length, SKI,l1::006BCC-10-10-AF, Diba industries, Danbury. CT 06810, US) is loaded with 200 mg:of Nuchar HD activated carbon (MeadWeStYaco Corporation. Richmond, VA, USA) Slurried in water. The column is:
packed by flowing water through it, which results in a packed column volume of 0:,:g niL
1001301 The activated: carbon column is pre-qui librated with about 10 CV of 25 mM
Tris at pH 7..(). Virus :spiked MAB feed is pumped through the activated carbon column at I mUmin (0,8 CV/min) with Watson Marlow cassette pumps. 2 ml fractions' arc collected from the effluent at column loads of 0,2 kg/L .0=5 kg1I,, mid 1.0 kg/L. The samples are titrated using a virus infectivity assay, as described in Example, 4.
[00131] As summarized in Table VI and Figure 2, this :experiment demonstrates that greater than 5,0 1,,RV of retroviruses and retrovirus-like particles, represented. by XMuLV, are removed from a solution of MABII by flowing the solution though a column of activated carbon at column loads of 0.2 41.I.õ 0.5.14/1õ and 1,0 kg/L. The results indicate that activated Carbon can betsed to Selectively remove retroviruses and retroVirus-like particles from a MABII monoclonal antibody solution, Table VI. 1,1-ZY of XMLILV removed from1MA Bii in fractions: collected at various column loads of MABII on the activated carbon column. Note that the concentration of XMULV in the fractions is below the levels.of detection.
Column load of:M.A./Ill LIZ.V of Xl\ttuiLV
on activated carbon from MABI.1 .(kg/Li 0.2 0.5 >5.54 1.0 >5 54 Example 11. Method to determine whether activated carbon can he used to selectively remove retrovirilses and/or retroviros-like particles from a MA Bill monoclonal antibody solution f001321 This is a third representative example along with Example 9 and Example demoiistrata method to determine if retrOvirozies and retro virus4ike particles, represented by XMul,V, are removed from a solution containing a monoclonal antibody (Lg., MABIII) by flowing through a column of activated carbon. The results indicate that activated carbon can be used to selectively remove retroyitwes and retroVirns-like particles froinaMABIII monoclonal antibody solution.
[00133] :A SolutiOn of MABEE is spiked With.:XMIILY and flowed through a column of activated carbon, as described below.
[00134] MABIII is, provided by Merck. Sewn aS a clarified cell culture. It is captured using Protein A chromatography (eluting with 20 naM glycine-hydrocbloride buffer at OH 2.4 The eluate has high levels of HCP and is therefore purified a second time using Protein A column chromatography. Belbre loading the chromatography column, the sodium chloride concentration of the MA.BUT
solution is increased to 0.5 M. The MA.B1.11 antibody is then eluted with 25 mM acetic acid, 25 rnM glyeine HC1. The pH of the MAR11.1 eluate is adjusted to pH 7.0 with 1 M
Iris base and the solution is filtered through a Stericup-GP 022 um Mi llipore Express PLUS membrane (1 L, catalogue number: SCGPLIO2RE, EMD Millipore Corporation, Billerica, MA, 01821, USA). The feed is dialyzed into 25 mM Tris pH 7.0 twice with dialysis tubing (Standard RC Dialysis Trial Kits; Spectra /Por 1-3, 1.5K M
WCO, 54 111111 FLAT WIDTH, serial number: 132725, Spectrum Laboratories, Inc. Rancho Dominguez, CA, 902:20 USA). The solution is filtered through a Stericup-GP
0.22 Express PLUS membrane (1 L, Catalogue number: SCGPUO2RE, .EMI) Millipore Corporation, Billerica, MA, 01821, USA), 1001351 MAB UI teed at pH 7.0:is spiked with Ultrapure grade of XMIILV that is produced according to the procedure described in Example 3 to a target spike of 1.0E+06 TCID50/mL. The spiked feed is filtered through a 0.45 um Durapore filter before contacting with activated carbon.
[00136] A glass chromatography column (Omni fit Benchmark Column 10 nun/100 mm, 10 mm diameter, 100 mm length. SKU: 006BCC-10-10-AF, Diba industries#
Danbury. CT 06810, US) is loaded with 200 mg of Nitchar RD activated carbon (MeadWestVaco Corporation, Richmond, VA,, USA) slurried in water. The column is packed by flowing water through it, which results in a packed column volume of 0.8 mL.
[00137] The activated carbon column is pre-equilibrated with about 10 CV of 25 niM iris at pH 7Ø Virus spiked MAB feed is pumped through the activated carbon column at 1 ml.,/rnin (0,8:04/min) with Watson Marlow cassette pumps. 2 ml fractions .arc collected in the effluent at column loads:of 0.2 kg/L, 0.5 kg/L, and 1.0 .kg/L. The samples are titrafed using a virus infectivity assay, as described in Example 4, 100138] As summarized in Table VII and Figure 2, this experiment demonstrates that greater than 5.0 LRV ofretroviruses and retrovirus-like particles;
represented by X.MuLV,:are:reinoved frorriA solution ofM.A.B111. by flowing the solution though a column of activated carbon at column loads, of 0.2 kg/L:03 kg/.L and 1.0 kg/L.
The results indicate that activated carbon can be used to selectively remove retroviruses and retrovirus,like particles from a MA131111 monoolobal antibody solution.
Table VII. LRV of XMuLV removed from MAI3III in fractions collected at variOtts column loads of MABIll on an activated carbon cOlumn. Note that the concentration of XiMuLV in the fractions is below the levels of detection.
Column load of MAB111 LRy of XMoLV from on activated carbon MA:131111 (kg/L) 0.2 0.5 LO ?5.10 Example 12. Method to determine whether activated carbon can be used to selectively remove retroviruses and/or retrovirus-like particles from a MABIV monoclonal antibody solution [001391 This is a forth representative example along with Example 9, Example 10, and Example 11 that demonstrates a method to determine if retroviruseS and retrovirus-like particles, represented by XMul...V, are removed from a solution Containing a Monoclonal antibody (Le., MABIV ) by flowing through a column of activated carbon, The results indicate that actiVated caxbon can be used to selectively remove retroviruses and retrovirus-like particles from a MAB1V monoclonal antibody solution.
[00140] A: solution of MAB1V is spiked with XMULV and flowed through a column of activated carbon, as described below:
[001411 MABIV monoclonal antibody is obtained from Merck Serono :Biodevelopment as a:10 git aqueous solution containing10 tnM citric acid, 100 mI\4 glyeine, 100mM sodium chloride, and 0,01%. of Tween, The feedis then dialyzed into 25 mM =Tris 7:0 twice with dialysis tubing (Standard RC Dialysis Trial Kits, SpectraTor 1-3, 3,5K,MWCO, 54 ram FLAT WIDTH, serial number: 1.32725, Spectrum Laboratories Inc., Rancho Domingue4, (.7A, 90220 USA). The solution is filtered through a Sterieup-GP 0.22 p.m Millipore Express PLUS membrane (1 L, catalogtte number; SCOPUO2RE, E MD Millipore Corporation, Billerica, MA, 01821, USA). MABIV feed at pH 7.0 is spiked. with Ultrapure grade of '..5(MuLV that is produced according to the procedure described in Example '3'W a target spike of 1.0E+06 Tcm,frimL. The spiked feed is filtered through a 0.45 Durapore filter before contacting with activated carbon.
[00142] A glass chromatography column (Omnifit Benchmark Column 10 mm! 00 mm, 10 min diameter, 100 mm length, SKU: 006BCC-1.0-10-AF, Diba Industries, Danbury, CT 06810, US) is loaded with 200 mg of Nuchar HD activated carbon (MeadWestNraco Corporation, Richmond, VA, USA) slurried. in water. The column is packed by flowing water through it, which results in a packed column volume of 0,8 tuL.
[001431 The activated carbon column is 07e-equilibrated with about 10 CV of 25 inl\.4 iris at pH 7,0. Vitus spiked MAB feed is pumped through the activated carbon column at 1 mi.,./min (0.8 CV/min) with Watson Marlow cassette pumps. 2m1 fractions are collected, from the effluent at column toads of 0.2 kg/I_, 0.5 kg/L, and 1,0 kg/L. The samples are titrated using u virus infectivity aSSay, as described in Example 4.
1001441 As summarized in Table VIII and Figure 2, this experiment demonstrates that greater than 5.0 1_,KV of retroviruses and retrovirus-like particles, represented by 004111...V, are removed from a solution Of MA.131V b flowing the solution though a.
column of activated carbon using at whin-in:Wads of 02 0.5 kg/h, and 1.0 kg/I¨
The results indicate that activated.earbon can be used to selectively remove retroviruseS and retrovirus-like particles from a WIA131V monoclonal antibody solution.
Table VIII. IRV of XMULV temOved from MAIIIV in fractionscolteeted at various column loads of MABIV oua activated carbon column. The exmlinellt was run in duplicate and the value listed is an average ofthose two values. Note that the concentation of NMI.t.IN in the fractions, bekliv the levels of detection.
Column load,of MABJV
L.RV of X:MuLY from on activated carbon MAMV
(kga.) 0,2 >5,29 0.5 25.29 1.0 [00145] The specification is most thoroughly understood in light of the teachings' of the references cited within the:Specification which are hereby incorporated by reference. The embodiments within the specification provide an illustration of embodiments in this invention and should not be construed to limit its scope.
The skilled artisan readily recognizes that many other embodiments are encompassed by this invention: All publications and inventions are incorporated by reference in their entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supercede any sach material. The citation of any refetenees herein is not an admission that such A-:ferentes are prior art to the present invention.
[00146] Unless otherwise indicated, all numbers expressing quantities of ingredients, cell culture, treatment conditions, and so thrth used in the specification, including claims, are to be understood as being modified in all instances by the term "about" Accordingly, unless otherwise indicated to the contrary, the numerical parameters are approximations and may vary depending upon the desired properties sought to be obtained by the present invention. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer re every element in the scries. Those ,skil led in theart Will recognize; or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention de.scribed herein. Such equivalents are intended to be encompassed by the following claims.
[00147] Many modifications and variations of this invention:can be made without departing from. its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein areoffered by way Of eNample only and are not meant to be limiting in any way. it is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims (17)

1. A method for determing whether activated carbon can be used for the removal of viruses from sample containing a target protein, the method comprising the steps of:
(a) providing a portion of a sample comprising a target protein;
(b) adding a virus to the portion of the sample in the amount ranging from 10 to 10 9 PFU/mL;
(b) flowing the portion of the sample through a column packed with activated carbon;
(c) collecting one or more flow-through fractions from the column; and (d) measuring the virus amount in the one or more flow-through fractions, wherein a reduction in the virus amount in the one or more flow-through fractions by at least 3.0 LRV is indicative that activated carbon can be used for the removal of viruses from the sample.

2. The method of claim 1, wherein the sample comprises a protein concentration equal to or greater than 0.2 g/L.

3. The method of claim 1, wherein the sample comprises a protein concentration equal to or greater than 0.5 g/L.

4. The method of claim 1, wherein the sample comprises a protein concentration equal to or greater than 1 g/L.

5. The method of claim 1, wherein the sample comprises a pH in the range of 3.0-10.0 and/or a salt concentration less than 0.5M.

6. The method of claim 1, wherein the virus is selected front the group consisting of a retrovirus, a retrovirus-like particle, and a parvovirus.

7. The method of claim 1, wherein the virus is selected from minute virus of mice and xenotropic murine leukemia virus.

8. The method of claim 1, wherein the sample comprises a cell culture feed.

9. The method of claim 6, wherein the cell culture feed is a CHO cell culture feed.

10. The method of claim 1, wherein the target protein is a recombinant protein

11. The method of claim 1, wherein the target protein is a therapeutic protein.

12. The method of claim 1, wherein the target protein is an antibody.

13. The method of claim 10, wherein the antibody is a monoclonal antibody.

1.4. The method of claim 1, wherein the sample comprises a. clarified cell culture feed,

15. The method of claim 14, wherein clarified cell culture feed is obtained using .one or more methods selected from centrifugation, settling, depth filtration, screen filtration, flocculation, use of a stimulus responsive polymers and pH
change.

16. The method of claim 1, wherein the sample comprises an eluate obtained from a Protein A chromatography column, wherein the eluate comprises the target protein.

17. The method. of claim 1 wherein step (a) employs one or more methods selected from the group consisting of 50% tissue culture infectious dose (TCID50) assay, plaque-forming unit assay, focus-forming unit assay, lethal.
dose 50% assay, hemagglutination assay, fluorescent focus assay (FFA), bicinchoninic acid assay (BCA), single radial immunodiffusion assay (SRID), quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay. (ELISA), and transmission electron microscopy.